The details of the mechanism which transfers mammalian mitochondrial DNA (mtDNA) from mother to offspring and the constraints which this mechanism would place on the origin of mitochondrial genetic diversity are currently unknown. The frequent occurrence of mtDNA sequence polymorphisms within a species suggests that simple transfer of a female's mitochondrial complement to her offspring does not occur and that a more complex path is followed which transfers only a small subset of the maternal mitochondria to the new embryo. Passage through this bottleneck would allow for the rapid appearance of new mitochondrial genotypes in the population. To test this model, heteroplasmic mice (which contain two different mtDNA genomes) were constructed by embryo fusion at the single cell level. Analysis of offspring of heteroplasmic female mice indicate both mtDNA species are transmitted to offspring; they appear to be transmitted at different levels in different progeny. Experiments are described which will determine if the segregation rates of the two mtDNA species in different nuclear gene backgrounds are identical, and whether both the level of overall mtDNA heteroplasmy and possible tissue differences in the levels of heteroplasmy occur. In addition, the rates at which the two different mtDNA species segregate in cell lines derived from these animals will be determined. Finally, a sensitive test for the presence of paternal mtDNA is described. These experiments will help define the kind and amounts of mtDNA variation that can occur in mammals. A second major goal of this proposal is to develop methods for mtDNA transformation. Mitochondria from various sources will be transferred into embryos, from somatic cells of various types. Once this is accomplished, mtDNA introduction into the organelle will be attempted using chemical treatment, liposome fusion, electroporation, or use of a particle accelerator to transform either cell lines or isolated mitochondria with exogenous mtDNA. These experiments will allow the construction of animals carrying in vitro mutated mtDNAs, and permit an examination of the role of mtDNA function in muscle and nerve myopathies. These animals and the techniques that allow their construction should also help in understanding cytoplasmic movement during early embryo development, movement of cell lineages, and the changes in mitochondria which occur during early development.